US5296116A - Capillary electrophoresis using time-varying field strength - Google Patents

Capillary electrophoresis using time-varying field strength Download PDF

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US5296116A
US5296116A US07/900,223 US90022392A US5296116A US 5296116 A US5296116 A US 5296116A US 90022392 A US90022392 A US 90022392A US 5296116 A US5296116 A US 5296116A
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time
electric field
electrophoresis
capillary
separation
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Andras Guttman
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Beckman Coulter Inc
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Beckman Instruments Inc
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Assigned to BECKMAN INSTRUMENTS, INC. reassignment BECKMAN INSTRUMENTS, INC. ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: GUTTMAN, ANDRAS
Priority to JP6501814A priority patent/JPH07508096A/ja
Priority to PCT/US1993/005809 priority patent/WO1993025899A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis

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  • the present invention relates to electrophoresis, particularly capillary electrophoresis, and more particularly capillary gel electrophoresis.
  • Cantor et al [13] Cantor, C. R.; Smith, C. L.; Mathew, M. K.; Ann. Rev. Biophys. Biophys. Chem., 1988, 17, 287-304) introduced the pulsed field method (changing the direction and magnitude of the field in an oscillating manner), that takes advantage of the elongated and oriented configuration of large DNA ( ⁇ 50 kbp) molecules in gels.
  • Heiger et al [6] described the capillary gel electrophoretic separation of double-stranded DNA molecules up to 23,000 base pairs in size using the pulsed field technique with very low gel concentrations.
  • the present invention is directed to a technique that provides enhanced separation resolution in electrophoresis.
  • This technique involves the use of a time-varying field strength, which may be progressively increasing or decreasing, constantly or otherwise, as a function of time.
  • the shape of the field strength with respect to time may be continuous or stepwise over time, monotonic or otherwise.
  • FIG. 1 is a schematic diagram of an automated electrophoresis instrument.
  • FIG. 2 compares electropherograms showing separation of a ⁇ X174 DNA restriction fragment mixture by capillary gel electrophoresis using different constant applied electric field (isoelectrostatic).
  • A 100,
  • B 200,
  • C 500 V/cm.
  • FIG. 3 is an electropherogram showing separation of a ⁇ X174 DNA restriction fragment mixture by capillary polyacrylamide gel electrophoresis using an increasing voltage. Dotted line represents the current output.
  • FIG. 4 is an electropherogram showing separation of the a ⁇ X174 DNA restriction fragment mixture by capillary polyacrylamide gel electrophoresis using decreasing voltage gradient. Dotted line represents the current output.
  • FIG. 5A is an electropherogram showing separation of a pBR322 DNA restriction fragment mixture by capillary polyacrylamide gel electrophoresis using an increasing stepwise gradient field;
  • FIG. 5B represents current output.
  • FIG. 6 is a table showing the theoretical plate number values of the peaks on FIGS. 2-4, calculated by using the System Gold Software Package (Beckman Instruments, Inc., Fullerton, Calif.).
  • the present invention is described below in reference to separation of DNA restriction fragments in capillary electrophoresis, and in particular capillary gel electrophoresis. However, it is understood that the technique of the invention can be to apply to other types of electrophoresis (e.g. slab gel electrophoresis) and for other types of samples (e.g. proteins, peptides).
  • electrophoresis e.g. slab gel electrophoresis
  • samples e.g. proteins, peptides.
  • the P/ACETM System 2100 capillary electrophoresis apparatus (Beckman Instruments, Inc., Fullerton, Calif.) was used. Said system 10 is schematically shown in FIG. 1. The details of the system have been omitted since the system is publicly available.
  • the capillary column 12 is encased in a cartridge 14 which is supported to allow the ends of the capillary to access vials 16 and 18 of electrolyte (liquid or gel) or sample solutions.
  • Capillary referred herein means tubing having inside diameter typically less than 1000 ⁇ m and more typically less than 300 ⁇ m. Coolant is pumped through the cartridge interior for controlling the temperature of the capillary 12.
  • a detector 20 is provided to detect separated species.
  • the vials are carried on carousels 22 and 24 which are rotated by motors 26 and 28 to position selected vials at the ends of the capillary 12.
  • a selected solution can be forced into the capillary by submerging one end of the capillary into the solution and gas pressurizing the vial by conventional means.
  • a low concentration polymerized gel i.e., polymer network
  • the gel in the capillary can be replaced by applying a rinse operation mode of the system, whereby the gel in the capillary can be flushed out of the capillary and a fresh gel can subsequently fill the capillary.
  • the system 10 has a sample injection mode which injects sample from a vial into one end of the capillary by either electromigration or gas pressure injection. Electrodes 26 and 28 are provided to apply the required high voltage (in the order of several hundred volts per cm of capillary) from voltage supply 34 for electromigration injection as well as for carrying out electrophoresis. Electrophoresis is performed with the two ends of the capillary dipped into electrolyte containing vials.
  • the electrolyte can be in the form of buffer solution similar to the buffer used in the process of forming the gel, or in the form of gel (i.e. a gel buffer system).
  • the operation and sequence of various functions of the system are carried out automatically under the control of a controller 36 programmable by the user. These functions include applying a time-varying electric field across the electrodes 26 and 28 in accordance with a user programmed profile such as those described below.
  • the cathode is on the injection side and the anode is on the detection side. Therefore, the negatively charged DNA molecules migrate toward the anode in the gel filled capillary column.
  • the separations were monitored on column at 254 nm.
  • the temperature of the capillary column was kept constant at 20° C.,+/-0.1° C., by the liquid cooling system of the P/ACETM instrument.
  • the electropherograms were acquired and stored on an Everex 386/33 computer.
  • the capillary used is packed with gel as a separation support medium.
  • ⁇ X174 DNA Hae-III digest and the pBR322 DNA Msp-I digest restriction fragment mixtures were diluted with deionized water to a concentration of 25 ⁇ g/ml before injection, and were stored at -20° C.
  • Ultra pure grade acrylamide, Tris, boric acid, EDTA, ammonium persulfate and tetramethylethylenediamine (TEMED) were used in the experiments (Schwarz/Mann Biotech, Cambridge, Mass.). All buffer and acrylamide solutions were filtered through a 0.2 ⁇ m pore-size filter (Schleicher and Schuell, Keene, N.H.) and carefully vacuum degassed.
  • the total length of the gel-filled capillary column was 470 and 670 mm (400 and 600 mm to the detection point), respectively.
  • the samples were injected by the pressure injection mode of the P/ACETM system, typically 5 sec, 0.5 psi. Estimated injection amount: 0.1 ng DNA.
  • Time-varying field strength profiles were programmed in the P/ACETM instrument with continuously increasing or decreasing voltage separation modes.
  • stepwise time-varying field separation mode constant voltages were used for different time periods as specified in the corresponding FIG. 5B.
  • FIG. 2 compares the separations of a ⁇ X174 DNA Hae-III restriction fragment mixture using different constant field strengths. Conditions: replaceable polyacrylamide gel column, effective length to detector 40 cm, total length, 47 cm; buffer, 0.1M Tris-borate, 2 mM EDTA (pH 8.35).
  • FIG. 2A shows the electropherogram 37 of the test mixture when 100 V/cm electric field is applied to the gel-filled capillary column. At this low field strength DNA molecules act more like random coils, so good separation can be achieved even for fragments above 10 3 base pairs (sieving effect [18]). This separation of the larger fragments is attained at the cost of longer separation time (>50 min). However, some of the smaller fragments are not fully resolved.
  • FIG. 2A shows, there is an incomplete separation between peak 46 and peak 47 (271 - and 281-bp fragments).
  • Similar results have been attributed by Karger and co-workers [6] to diffusional band broadening due to the long separation time in very diluted gel filled capillary columns. By increasing the applied electric field to 200 V/cm, complete separation of all eleven fragments is achieved in 27 min. (see electropherogram 38 in FIG. 2B).
  • Equation 1b states that the actual velocity, v(t) of a DNA molecule is influenced by the field strength in use at a given time and by the mobility, which is also a function of the field strength.
  • the electrophoretic acceleration (a) can be expressed as the change in electrophoretic velocity, i.e. the product of the electrophoretic mobility and the field strength at a given time:
  • Peak efficiency (theoretical plate value N) and resolution (R S ) are also affected by the momentary field strength ([22] Karger, B. L.; Cohen, A. S.; Guttman, A. J. Chromatogr. 1989, 492, 585-614.)
  • the change in the theoretical plate number N is a linear function of the acceleration, ##EQU2## where L is the effective length of the capillary and D is the diffusion coefficient of the solute.
  • the change in resolution is proportional to the square root [22] of the acceleration, dR S /dt ⁇ d(a 1/2 )/dt when a linearly time-varying field strength is used.
  • Time-varying field strength can be used in increasing, decreasing, constant or otherwise, continuous or stepwise modes or in any combination thereof, if necessary.
  • the fluctuation of the field strength in case of a profile involving a combination of increasing and decreasing field strength
  • Pulsed field electrophoresis uses oscillating field of at least several cycles per second (e.g. 50 Hz).
  • FIG. 3 is an electropherogram 56 which shows a separation of the ⁇ X174 DNA restriction fragment mixture using constantly increasing electric field strength over time (0 V/cm to 400 V/cm in 20 min). Other conditions remain the same as before. Because the capillary separation channel has uniform internal diameter, the current varies directly with voltage. It is easier to represent the time-varying voltage by showing the time-varying current in FIG. 3 as a dotted line 58. Full separation of all the test mixture components was achieved in less than 19 min. As FIGS. 3 and 6 show, the apparent efficiency (i.e., theoretical plate number [7]) of the last several peaks (49, 50 and 51) is greater compared to FIG. 2B, where full separation of all the sample components was also attained.
  • the apparent efficiency i.e., theoretical plate number [7]
  • the applied electric field can be decreased over time, as shown in FIG. 4.
  • a constantly decreasing field strength gradient of 400 V/cm to 100 V/cm in 20 min (dotted line 59 in FIG. 4) was applied to the linear polyacrylamide network-filled capillary column.
  • Other conditions remain the same.
  • baseline separation of all eleven components of the ⁇ X174 restriction fragment mixture was achieved in less than 10 min, which is comparable to the separation time shown in FIG. 2C.
  • the larger fragments migrated past the detector window slower due to the lower field strength at the end of the separation. This causes an apparent loss in efficiency or theoretical plate number N (equation 9, see table in FIG. 6) and resolution, particularly for the last three peaks.
  • FIG. 5A shows an electropherogram 57 which the separation of a pBR322 DNA restriction fragment mixture employing a stepwise time-varying voltage method in a longer capillary column (effective column length: 60 cm).
  • the method consisted of three consecutive steps, 100 V/cm from 0 to 40 min., 200 V/cm from 40 to 70 min. and 400 V/cm from 70 to 100 min. (see current profile 60 in FIG. 5B). Other conditions remain the same. In this way, full separation of almost all the components was attained.
  • FIG. 5A shows an electropherogram 57 which the separation of a pBR322 DNA restriction fragment mixture employing a stepwise time-varying voltage method in a longer capillary column (effective column length: 60 cm).
  • the method consisted of three consecutive steps, 100 V/cm from 0 to 40 min., 200 V/cm from 40 to 70 min. and 400 V/cm from 70 to 100 min. (see current profile 60 in FIG. 5B). Other conditions remain the same. In
  • a simple time-varying field strength method was introduced in order to increase the resolving power in capillary polyacrylamide gel electrophoresis separation of DNA restriction fragment mixtures.
  • the use of increasing, decreasing, continuous or stepwise voltage techniques showed that the resolving power can be optimized for a given DNA chain length range, and separation time can be significantly reduced.
  • the best separation with minimum time requirement was achieved by using a continuously decreasing applied electric field. It is important to note that with the use of field strength gradient methods, the apparent peak efficiency and resolution may be misleading since the different size components migrate past the detector window with a velocity that is determined by the voltage in use at that point in time.
  • Other types of time-varying parameters may be employed, such as current, power and temperature and the combination of those can also be used to optimize capillary gel electrophoretic separations of a given sample mixture.

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Cited By (14)

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US5512158A (en) * 1995-02-28 1996-04-30 Hewlett-Packard Company Capillary electrophoresis method and apparatus for electric field uniformity and minimal dispersion of sample fractions
US5985120A (en) * 1997-06-12 1999-11-16 University Of Massachusetts Rapid analysis of analyte solutions
US6001231A (en) * 1997-07-15 1999-12-14 Caliper Technologies Corp. Methods and systems for monitoring and controlling fluid flow rates in microfluidic systems
US6372106B1 (en) * 1999-07-26 2002-04-16 Applera Corporation Capillary electrophoresis method and apparatus for reducing peak broadening associated with the establishment of an electric field
US20060074186A1 (en) * 2004-01-16 2006-04-06 Northwestern University Sparsely cross-linked nanogels: a novel polymer structure for microchannel DNA sequencing
US20090136932A1 (en) * 2007-03-16 2009-05-28 Craighead Harold G Fibers with isolated biomolecules and uses thereof
US20200249135A1 (en) * 2015-07-10 2020-08-06 Picometrics Technologie System for concentration and pre-concentration by sample stacking and/or purification for analysis
US10996212B2 (en) 2012-02-10 2021-05-04 The University Of North Carolina At Chapel Hill Devices and systems with fluidic nanofunnels for processing single molecules
US11053535B2 (en) 2011-09-12 2021-07-06 The University Of North Carolina At Chapel Hill Devices with a fluid transport nanochannel intersected by a fluid sensing nanochannel and related methods
US11067537B2 (en) 2013-03-13 2021-07-20 The University Of North Carolina At Chapel Hill Nanofluidic devices for the rapid mapping of whole genomes and related systems and methods of analysis
US11073507B2 (en) * 2013-02-28 2021-07-27 The University Of North Carolina At Chapel Hill Nanofluidic devices with integrated components for the controlled capture, trapping, and transport of macromolecules and related methods of analysis
US11656220B2 (en) 2016-09-08 2023-05-23 Hemex Health, Inc. Diagnostics systems and methods
US11701039B2 (en) * 2016-09-08 2023-07-18 Hemex Health, Inc. Diagnostics systems and methods
US11740203B2 (en) 2019-06-25 2023-08-29 Hemex Health, Inc. Diagnostics systems and methods

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EP1929314B1 (fr) * 2005-09-22 2018-12-19 Koninklijke Philips N.V. Accelerometre adaptatif bidimensionnel a dielectrophorese
EP2425216B1 (fr) 2009-04-27 2019-10-23 Expedeon Holdings Ltd. Systèmes et procédés à filtres réjecteurs électrophorétiques programmables

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US5512158A (en) * 1995-02-28 1996-04-30 Hewlett-Packard Company Capillary electrophoresis method and apparatus for electric field uniformity and minimal dispersion of sample fractions
US5985120A (en) * 1997-06-12 1999-11-16 University Of Massachusetts Rapid analysis of analyte solutions
US6001231A (en) * 1997-07-15 1999-12-14 Caliper Technologies Corp. Methods and systems for monitoring and controlling fluid flow rates in microfluidic systems
US6221226B1 (en) 1997-07-15 2001-04-24 Caliper Technologies Corp. Methods and systems for monitoring and controlling fluid flow rates in microfluidic systems
US6616823B2 (en) 1997-07-15 2003-09-09 Caliper Technologies Corp. Systems for monitoring and controlling fluid flow rates in microfluidic systems
US7169276B2 (en) 1999-07-26 2007-01-30 Applera Corporation Capillary electrophoresis method and apparatus for reducing peak broadening associated with the establishment of an electric field
US6372106B1 (en) * 1999-07-26 2002-04-16 Applera Corporation Capillary electrophoresis method and apparatus for reducing peak broadening associated with the establishment of an electric field
US20020100689A1 (en) * 1999-07-26 2002-08-01 Pe Corporation (Ny) Capillary electrophoresis method and apparatus for reducing peak broadening associated with the establishment of an electtric field
EP1455183A2 (fr) * 1999-07-26 2004-09-08 Applera Corporation Procédé et appareil permettant de réduire une largeur de pic associe a l'etablissemant d'un champ electrique
EP1455183A3 (fr) * 1999-07-26 2004-12-29 Applera Corporation Procédé et appareil permettant de réduire une largeur de pic associe a l'etablissemant d'un champ electrique
US20090011420A1 (en) * 2004-01-16 2009-01-08 Northwestern University Sparsely cross-linked nanogels: a novel polymer structure for microchannel dna sequencing
US7399396B2 (en) 2004-01-16 2008-07-15 Northwestern University Sparsely cross-linked nanogels: a novel polymer structure for microchannel DNA sequencing
US20060074186A1 (en) * 2004-01-16 2006-04-06 Northwestern University Sparsely cross-linked nanogels: a novel polymer structure for microchannel DNA sequencing
US7531073B2 (en) 2004-01-16 2009-05-12 Northwestern University Sparsely cross-linked nanogels: a novel polymer structure for microchannel DNA sequencing
US20090136932A1 (en) * 2007-03-16 2009-05-28 Craighead Harold G Fibers with isolated biomolecules and uses thereof
US20100331196A1 (en) * 2007-03-16 2010-12-30 Cornell University; Cornell Center for Technology Enterprise and Commercialization (CCTEC) Electron beam nucleic acid sequencing
US11053535B2 (en) 2011-09-12 2021-07-06 The University Of North Carolina At Chapel Hill Devices with a fluid transport nanochannel intersected by a fluid sensing nanochannel and related methods
US10996212B2 (en) 2012-02-10 2021-05-04 The University Of North Carolina At Chapel Hill Devices and systems with fluidic nanofunnels for processing single molecules
US11073507B2 (en) * 2013-02-28 2021-07-27 The University Of North Carolina At Chapel Hill Nanofluidic devices with integrated components for the controlled capture, trapping, and transport of macromolecules and related methods of analysis
US11067537B2 (en) 2013-03-13 2021-07-20 The University Of North Carolina At Chapel Hill Nanofluidic devices for the rapid mapping of whole genomes and related systems and methods of analysis
US11307171B2 (en) 2013-03-13 2022-04-19 The University Of North Carolina At Chapel Hill Nanofluidic devices for the rapid mapping of whole genomes and related systems and methods of analysis
US20200249135A1 (en) * 2015-07-10 2020-08-06 Picometrics Technologie System for concentration and pre-concentration by sample stacking and/or purification for analysis
US11656220B2 (en) 2016-09-08 2023-05-23 Hemex Health, Inc. Diagnostics systems and methods
US11701039B2 (en) * 2016-09-08 2023-07-18 Hemex Health, Inc. Diagnostics systems and methods
US11740203B2 (en) 2019-06-25 2023-08-29 Hemex Health, Inc. Diagnostics systems and methods

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